Streptococccus mutans is a ubiquitous oral bacterium identified as a prominent etiologic agent of human dental caries. Certain strains of S. mutans have also been identified as causative agents of bacterial endocarditis, discovered within atherosclerotic plaque, and demonstrated to invade human coronary epithelial cells. The health care cost associated with dental decay stemming from S. mutans and other cariogenic organisms alone is enormous, over $64 billion annually in the USA. This project addresses membrane biogenesis and protein secretion in S. mutans. Membranes comprised of a semi-permeable lipid bilayer enclose all cells and, by dictating what can and cannot cross by virtue of proteins embedded within them, determine cellular function. S. mutans shares features common to bacterial membrane protein insertion and protein secretion systems common to the most widely studied model organism Escherichia coli, as well as to those of mitochondria and chloroplasts, but it exhibits multiple unique properties as well. For this reason it is gaining in reputation as a model organism to better understand the protein translocation machinery in other streptococci, and Gram-positive bacteria in general. All known virulence properties of S. mutans are a consequence of its membrane protein composition and/or secreted extracellular proteins. Since biological membranes are composed of ~50% proteins by mass, and these represent most known and existing drug targets, a better understanding of the membrane insertion and transport pathways of S. mutans that guide its virulence factors to their necessary locations will facilitate future targeted therapies against this and related pathogens. This projec focuses on two critical co-translational translocation pathways, the signal recognition particle (SRP) pathway found in all living cells, and the YidC insertase pathway found in bacteria/mitochondria/chloroplasts. The presence in Gram-positive bacteria of an additional SRP component called YlxM, and of dual YidC paralogs of differing function, was discovered in S. mutans. Additional components unique to S. mutans appear to exist as well. In this project, a combination of membrane proteomics, directed genetic, biochemical, and biophysical approaches, and complementary in vivo and in vitro analyses will improve the understanding of membrane protein insertion and secretion pathways of S. mutans and will identify specific membrane-localized substrates related to the pivotal virulence properties of competence development and mutacin production since secretion pathway mutants are defective in this regard. These studies are facilitated by the establishment of methodologies to purify transitionally active ribosomes from S. mutans, to assess membrane protein insertion in vivo and in vitro, and to utilize biolayer interferometry, co-immunoprecipitation, and chemical cross-linking to detect and measure interactions of membrane-localized secretion pathway components that are crucial to the transport and insertion of the substrates identified as part of this research.